Fungal degradation and bioremediation system for creosote-treated wood

ABSTRACT

A method for degrading and/or bioremediating waste wood containing creosote using a fungal inoculum is disclosed. The fungal inoculum comprises of at least one creosote-tolerant fungi, a lignocellulosic substrate and a nutrient supplement. The fungal inoculum is applied to the waste wood and maintained in an aerated and hydrated environment having temperature conditions sufficient to allow the inoculum to grow and metabolize the creosote. The inoculum and the waste wood are combined until an end product is achieved that is at least partially remediated or of a reduced volume.

BACKGROUND OF THE INVENTION

The present invention relates to fungal inocula and their use indegrading and bioremediating wood treated with chemical preservatives.More specifically, this invention discloses and claims a fungalinoculum, the method of its preparation, and its use in degrading andbioremediating wood treated with creosote.

Wood used in the construction of today's decks, docks and buildings, oras utility poles and railroad ties, is typically treated with a chemicalpreservative to prevent its deterioration and extend its service life.The chemical preservative used will generally depend upon the intendeduse of the wood and often includes chemicals such as creosote, chromatedcopper arsenate (CCA), ammoniacal copper quat (ACQ), andpentachlorophenol. For heavy timbers, poles, piles and railroad ties,creosote is commonly used because of its minimal cost and its relativeinsolubility in water, reduced volatility and high toxicity towood-destroying organisms.

The disposal of creosote treated wood once it reaches the end of itsuseful life requires careful consideration because of its toxicity. Forexample, creosote treated wood is generally not burned in open fires oropen stoves, fireplaces or residential boilers because of the toxicchemicals which may be produced as part of its smoke and ash.Accordingly, creosote treated wood is typically collected and stored atlandfills or other facilities. The amount of waste wood being stored atthese landfills or storage facilities is accumulating at an alarmingrate such that their collection and storage creates an environment wherethe contamination of the surrounding soil and groundwater by toxic,environmentally-persistent chemicals is a likely result.

Contamination of soils and groundwater with toxic,environmentally-persistent chemicals is a serious problem. Toxic,environmentally-persistent chemicals are those that are resistant todegradation in the natural environment. As such, these chemicals pose amulti-faceted problem in that as they persist and accumulate in theenvironment, their toxicity, including in many instances, provencarcinogenicity, presents substantial health risks to both animals andhuman beings. Environmental contamination from creosote-treated wood isa specific concern in view of the volume of creosote-treated waste woodexpected to be removed from service and disposed of in the near future.

The prior art is replete with methods for degrading hazardous chemicals.However, this prior art is generally, and specifically, directed towardshalogenated aromatic compounds. Suggested treatment strategies includeincineration of the waste in commercial or industrial incinerators orboilers under state and federal regulation, removal and isolation of thecontaminated materials, and degradation of the pollutant by bacteria.

All of these strategies suffer from serious deficiencies. Incinerationis extremely expensive due to the required energy and safety expense andthe necessity of moving the contaminated material to remote locations.Incineration is also impractical because of the large quantities ofwaste which needs processing. Removal and isolation of the contaminatedmaterial is also expensive and does nothing to effect a long-termsolution. Degradation of the chemicals using bacteria has also provenineffective due to the bacteria's specificity for particular chemicalsand its sensitivity to the toxic chemicals and environmental conditions.

U.S. Pat. No. 5,476,788 employs another strategy which utilizes aninoculum containing the lignin-degrading fungal species Phanerochaetechrysosporium, Phanerochaete sordida, or Trametes hirsuta to remediatesolid materials, such as soils, sludge, sediments, and debris (e.g.,woods), contaminated with pentachlorophenol. The inoculum contains oneor more of the fungal strains and a lignocellulosic substrate, i.e.,sawdust. In its use, the inoculum is combined with thepentachlorophenol-contaminated material and the entire mixture isaerated and hydrated until the inoculum metabolizes thepentachlorophenol to a less toxic product. Typically, this less toxicproduct includes pentachloroanisole.

Although the above-identified bioremediation strategy provides a usefulmeans of reducing the pentachlorophenol content in various solidmaterials, it does not address the concerns associated with other woodpreservatives currently used in the world today. Moreover, it fails toprovide a working strategy for other types of fungal strains whichrequire a more specific and unique environment to effectively remediatechemical preservatives such as CCA, ACQ, creosote, or pentachlorophenolor degrade wood treated with these chemicals.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that it discloses a fungalinoculum, the method of its preparation, and its use in degrading and/orbioremediating wood treated with the chemical preservative creosote.

The disclosed fungal inoculum generally comprises of at least onecreosote-tolerant fungus, a lignocellulosic substrate, and a nutrientsupplement. The creosote-tolerant fungal strains are preferably selectedfrom the group consisting of Antrodia radiculosa (FP-103272-sp),Antrodia radiculosa (FP-105309-R) and Antrodia radiculosa (L-11659-sp)and Neolentinus lepideus (Mad-534). The lignocellulosic substrate ispreferably sawdust or wood chips. Other feasible substrates are ricestraw, corn stalks, and wheat straw. The nutrient supplement ispreferably selected from the group consisting of corn steep liquor,cornmeal and wheatbran.

In one preferred embodiment, the fungal inoculum is prepared by firstgrowing the creosote-tolerant fungus in dark, aerobic conditions, havinga relative humidity and a temperature sufficient to support fungalgrowth. The fungus is then combined with a homogenous matrix comprisingsterile water, the nutrient supplement, and the sterilizedlignocellulosic substrate, to form the fungal inoculum. The fungalinoculum is then allowed to mature by exposing the mixture to dark,aerobic conditions, at a relative humidity and in a temperature rangesufficient to allow the fungus to reach a confluent growth.

Waste wood containing creasote is remediated or degraded by inoculatingthe waste wood in the fungal inoculum. To inoculate the waste wood, thefungal inoculum is first spread over the waste wood until all of thewaste wood is covered. The waste wood and fungal inoculum mixture isthen aerated and hydrated for a time and under conditions sufficient toallow the inoculum to at least partially remediate the creasote and/ordegrade the waste wood to a desired degradation product. In someinstances, the degradation product may be capable of reuse in paper orwood composites, or simply have a reduced volume.

It is an object of the present invention to provide a method forbioremediating and/or degrading chemically treated waste wood to achievea product having a reduced volume and/or the capacity to be reused as awood fiber resource.

It is another object of the present invention to provide a fungalinoculum, and a method for preparing said fungal inoculum, which isuseful in bioremediating and/or degrading chemically treated waste wood.

It is another object of the present invention to provide a fungalinoculum, and a method for using the fungal inoculum, to degrade and atleast partially remediate waste wood chemically treated with creosote.

One advantage of the present invention is that the creosote-tolerantfungi utilized do not require genetic alteration to specifically grow inthe presence of creosote. Thus, the introduction of the fungi into theenvironment provides no new, non-naturally occurring organisms.

Another advantage is that the preparation and use of the fungal inoculumis fairly simple and utilizes agricultural waste products and wasteproducts from saw mills and urban chipping. These products have theadditional advantage of providing a quick and low cost food source forthe fungus, while having the added effect of stimulating rapid andextensive fungal growth, as well as providing a readily storable andtransportable solid matrix.

Another advantage is that the inoculum and its method of use areparticularly well suited for waste woods such as pressure-treated lumberfrom buildings, decks, utility poles and railroad ties. Specifically,the solid matrix of the fungal inoculum provides a wood environment forfungal growth which is similar to that of the waste wood. The fungalstrain is, therefore, readily adapted to the waste wood environment uponinoculation and does not require a period of adjustment beforedegradation and bioremediation begins.

These and other objects and advantages of the invention are readilyunderstood in view of the following detail description and examples.

DETAILED DESCRIPTION OF THE INVENTION

Waste wood containing creosote is degraded and remediated in accordancewith the present invention by inoculating the waste wood with a fungalinoculum comprising at least one creosote-tolerant fungus, alignocellulosic substrate and a nutrient supplement. The fungal inoculumis applied to the waste wood and maintained in an aerated and hydratedenvironment having temperature conditions and a moisture contentsufficient to allow the inoculum to grow and at least partiallyremediate the creosote or degrade the preservative treated wood. Theinoculum and the waste wood are combined until a degradation product isachieved that is either of a volume consistent with the desires of thepractitioner or capable of being recycled and used for paper or othercomposite woods.

Creosote-tolerant fungi according to the present invention are generallydefined as fungi capable of surviving and sustaining growth while beingexposed to creosote. In the preferred embodiment, the creosote-tolerantfungi include, without limitation, Antrodia radiculosa (FP-103272-sp),Antrodia radiculosa (FP-105309-R), Antrodia radiculosa (L-11659-sp) andNeolentinus lepideus (Mad-534). Most preferably, the fungus utilized iseither Antrodia radiculosa (FP-103272-sp) or Neolentinus lepideus(Mad-534). The creosote-tolerant fungi, however, may also include anyother creosote-tolerant fungus capable of degrading or bioremediatingthe treated waste wood. Preferably, the chosen fungus should provide atwo and one-half percent (2.5%) or more dry weight loss in the wastewood after about ten weeks of reaction time.

In the preferred embodiment, the creosote-tolerant fungi are naturallyexisting fungi and not genetically altered or conditioned to grow underspecific conditions or in the presence of a particular preservative. Itis anticipated, however, that one skilled in the art may use agenetically altered or conditioned fungus in accordance with the presentinvention. Genetically altered or conditioned fungi may include, but arenot limited to, any fungus modified to grow in the presence of aspecific nutrient supplement or food source.

In accordance with the Budapest treaty, the strains Antrodia radiculosa(FP-103272-sp), Antrodia radiculosa (FP-105309-R), Antrodia radiculosa(L-11659-sp) and Neolentineus lepideus (Mad-534) were deposited with theAgricultural Research Culture Center (NRRL), an International DepositaryAuthority located at 1815 North University Street, Peoria, Ill. 610604U.S.A., on Jul. 30, 1999, and given the accession numbers NRRL 30168,NRRL 30170, NRRL 30167, and NRRL 30172, respectively.

The lignocellulosic substrate serves as a long-term food source for thefungal inoculum as well as a matrix for its storage and handling.Generally, “lignocellulosic substrate” refers to a substrate havinglignin, cellulose, or a combination of both lignin and cellulose. In thepreferred embodiment the lignocellulosic substrate includes sawdust orwood chips, either alone or in combination, but may also include anysubstrate that is capable of sustaining the growth of the fungi. Otherlignocellulosic substrates may include, without limitation, agriculturalresidues such as rice straw, corn stalk, wheat straw, etc.

Sawdust or wood chips are preferred, however, for several reasons: (1)they provide a long-term food source for the fungus while providingfungal growth in a wood environment similar to the creosote-treatedwaste wood environment experienced during inoculation; (2) they permitthe production of a large quantity of fungi in a single container; (3)they provide a substrate for easily storing and transporting the fungi;(4) they provide a matrix for convenient and even distribution of thefungus at the inoculation site; and (5) they provide a low cost use of awaste product from saw mills.

The nutrient supplement is defined as a supplement for thelignocellulosic substrate which provides a food source that stimulatesrapid and extensive fungal growth beyond that obtained from thelignocellulosic substrate alone. The nutrient supplement may be of anyfood source which accomplishes the above stated goal and may differdepending upon the fungus selected. Preferably, however, the nutrientsupplement used is either a corn steep liquor, cornmeal or wheatbran.

Preferably, the fungal inoculum is prepared by first growing thecreosote-tolerant fungus, or fungi, in culture containing malt extractagar. The culture is then typically incubated for one to two weeks, oruntil a confluent fungal growth is achieved over the agar surface. Theincubation is best performed in dark, aerobic conditions, at a relativehumidity of about 70%, and at a temperature range from about 20° C. to35° C., and more preferably at a temperature range from about 27° C. to32° C.

After achieving a confluent fungal growth, the fungus, lignocellulosicsubstrate and the nutrient supplement are combined to form the fungalinoculum, or “seeding.” The lignocellulosic substrate is firstheat-sterilized and allowed to cool. The sterilized lignocellulosicsubstrate is then mixed with the nutrient supplement and sterile wateruntil a homogenous matrix is formed. The lignocellulosic substrate ispreferably combined with the sterile water at 2-3 volumes of water pervolume of substrate, while the nutrient supplement is added in a rangefrom about 1% to 5% per volume water. Liquid nutrient supplement shouldalways be added to the sterile water first and solid nutrient supplementshould always be mixed with the substrate before adding the sterilewater. This will ensure a homogenous matrix.

The homogenous matrix is gently mixed with the fungal culture andallowed to grow to form the final fungal inoculum. The mixture isallowed to grow for a time and under conditions which allow the fungusto reach confluent growth. Preferably, the mixture is grown in darkaerobic conditions, at a relative humidity of about 70%, and in atemperature range from about 20° C. to 35° C., and more preferably at atemperature range from about 27° C. to 32° C. Typically, confluentgrowth should occur within 4-8 weeks, but depends specifically upon thefungal volume introduced into the matrix, the lignocellulosic substrate,and the nutrient supplement. For example, a heavy fungal inocula withsawdust will shorten the period of fungal growth.

The fungal inoculum is ready for use or storage as soon as it hasreached confluent growth. If immediate use is desired, the inoculum canbe readily transported to the bioremediation and/or degradation sitewhere it is applied to completely cover the waste wood. In thealternative, if storage is desired, the fungal inoculum can be stored at4° C. The storage at 4° C. slows the fungal development and preventsovergrowth.

In one preferred embodiment of the present invention, large quantitiesof industrial inoculum can be produced either with large numbers of trayinoculum, or in large durable plastic bags with aeration patches toallow the appropriate airflow. Trays and bags can be transported easilyto the field sites and applied on the waste wood. Production of inoculumdirectly in the truck or truckload container is also a possibility.

In another embodiment, steam is an alternative source for sterilization.This is especially useful in pilot plants where steam is readilyavailable. In accordance with this embodiment, the steam provides bothsterility and moisture content for the lignocellulosic substrate. Afterbeing steamed, however, the substrate temperature must be cooled so asto avoid killing the fungi.

Once transported to the bioremediation/degradation site, the fungalinoculum is spread over the waste wood until all of the waste wood iscovered. Preferably the waste wood is heat sterilized prior toapplication so as to minimize other environmental factors which mayeffect the ability of the fungi to properly degrade or bioremediate thewaste wood. Such factors typically include highly competitive bacteriaor other fungi.

The creosote-containing waste wood and fungal inoculum mixture is thenaerated and hydrated for a time and under conditions sufficient to allowthe inoculum to at least partially metabolize the creosote and degradethe waste wood to a desired degradation product. The inoculated wastewood is maintained in a dark, aerobic environment, at a relativehumidity of about 70%, and in a temperature range from about 20° C. to35° C., and more preferably in a temperature range from about 27° C. to32° C. The inoculation environment must have ample air space to ensureproper growth and to allow proper oxygen flow. In the absence of properoxygen flow fungal growth will be hampered.

The degradation of the wood by the fungal inoculum will generally resultin a degradation product having either a reduced volume or a reducedconcentration of creosote. Such a product will provide a resourcecapable of reuse in paper or wood composites, or simply have a reducedvolume such that storage or further processing is minimized.

The present invention is further explained by the following exampleswhich should not be construed by ways of limiting the scope or spirit ofthe present invention.

EXAMPLES Example 1

Selection of Preservative-Tolerant Fungi

Fungal strains were collected after an extensive screening of the fungallibrary at the Center for Forest Mycology Research within the U.S.Department of Agriculture, Forest Products Laboratory. The screeningprocess was first initiated by searching the library for fungal strainscollected from specific wood products typically treated with woodchemical preservatives, i.e., utility poles, boats, decks, docks andrailroad ties. Other strains were isolated from wood samples collectedfrom the field plots of the USDA Forest Services, Forest ProductsLaboratory in Gulfport, Miss. and from Picnic Point in Madison, Wis. Theselected fungal strains were then retrieved and further analyzed todetermine their tolerance to CCA, ACQ, creosote and pentachlorophenol.

Chemical preservative tolerance was determined by application of a“choice test.” The choice in this case was whether a particular fungalstrain would grow towards a wood treated with a chemical preservative ortowards a non-treated wood, or both.

To perform the “choice test”, a freshly grown fungal malt agar disk (9mm) was placed in the center of a Petri dish (14 cm diameter) containing12 ml of water agar, as described in Leithoff et al., “Growth of thecopper tolerant ground-rot fungus Antrodia vaillantii on differentsubstrates,” The Int'l Research Group on Wood Preservation,IRG/WP95-10121, incorporated herein by reference. In this particularapplication, however, no glass ring was applied to the agar disk. Apreservative-treated wood sample (1.5 cm×3 cm) was then placed at oneedge of the Petri dish while a non-treated wood sample was placed at theopposite edge. The plates were then incubated at 27° C. and 70% relativehumidity (RH) for 14 days, wherein fungal growth was monitored.

At the end of 14 days, most fungal strains showed a primary growthpreference towards the non-treated wood with no growth towards thetreated wood. Some strains, however, showed growth preference towardsboth directions. Table 1 lists the strains which exhibited the greatestamount of growth towards wood treated with the chemical preservativecreosote. These fungal strains were considered creosote-tolerant andselected for use in later degradation and bioremediation studies.

TABLE 1 Fungi Selected by Choice Test for Tolerance to PreservativesIsolate Preservative Strain Name Designation Source Creosote Melanoporianiger MD278 FPL-MC Polyporus sp FP101605 s FPL-MC Crustoderma dryinumKropp62-CI FPL-MC Gloeophyllum subferrugineum FPRI-508 FPL-MCNeolentinus lepideus RLG 7891-s FPL-MC Phanerochaete sordida Kropp 36 T2FPL-MC Peniphora pseudopini TCS-13 FPL-MC Ceriporia spissa 12A4ck FPL-MC— UpK FPL Neolentinus lepideus Mad-534 FPL Meruliporia incrassataMad-563 FPL Antrodia radiculosa FP-103272-sp FPL-MC Antrodia radiculosaL-11659-sp FPL-MC Antrodia radiculosa FP-105309-R FPL-MC FPL-MC: ForestProducts Laboratory Center for Forest Mycology Research FPL: ForestProduct Laboratory

Example 2

Determination of Optimum Growth Conditions

The fungal strain Meruliporia incrassata (TFFH-294) was used todetermine the optimum growth conditions for fungal strains to be used infungal inocula effective in bioremediating and/or degrading chemicallypreserved wood. Fungal strain Meruliporia incrassata (TFFH-294) is astrain that was isolated from the USDA-FS Forest Products Laboratoryresearch plots in Gulfport, Miss. Merulipuria incrassata (TFFH-294) hasbeen deposited with NRRL and assigned accessian number NRRL 30165.

Optimum Temperature

Four temperature settings, 20, 27, 32 and 37° C. were studied todetermine their effect on fungal growth. Four disks of fungal culturewere inoculated onto malt extract liquid medium (Difco Bacto maltextract). After incubation for 12 days at the various temperatures, themycelia of the fungi were harvested on Whatman No. 1 filter paper, airdried and measured for biomass dry weight.

The results showed that the optimal temperature for fungal growth was ina range between about 27° C. and 32° C. Incubation at a highertemperature setting of 35° C. showed a substantial decline in growth, asdid incubation at a lower temperature setting of 20° C.

Optimal Light Conditions

Light effect on growth was studied under three settings, 24 hours oflight, 12 hours of light with 12 hours of darkness, and 24 hours ofdarkness. Fungal inoculant was grown in malt extract liquid medium asabove for a period of 21 days. Mycelia were then harvested, air driedand weighed to obtain biomass measurements.

The results of light effect are shown in Table 2. Fungi produced morecell mass under the complete dark growth condition than under either amixture of light/dark or complete light. Cell mass measurementsincreased 33% at 24 hours dark cycle then at 24 hours light cycle.

TABLE 2 Light Effect on TFFH-294 Growth Light condition Dry Weights, mg(±S.D.) 24 hours of light 66 ± 8 12 hours of light/dark 70 ± 6 24 hoursof dark  88 ± 13

Defined Liquid Medium

Fungal cultures were inoculated in Bailey media (Bailey et al.,“Cellulase (B-1,4-Gucan, 4-Glucanohydrolase) from Wood-Degrading FungusPolyporus Chweinitzii,” Fr. I. Purification Biochem. Biophys. Acta,185:381-391 (1969)), and BIII media (Kirk et al., “Production ofMultiple Ligninases by Phanerochaete chrysosporium: Effect of SelectedGrowth Conditions and Use of a Mutant Strain,” Enzyme Microb. Technol.,8:27-32 (1986)) to determine which medium provided better fungal growth.Fresh fungal cultures were grown on malt extract agar plates. Four agardiscs (9 mm) of complete fungal growth were removed and inoculated into25 ml growth medium in 125 ml flasks. The flasks were then keptstationary at 27° C. and 70% RH for 21 days. After 21 days, the myceliawere harvested for biomass measurements.

The biomass measurements indicated that the biomass growth for TFFH-294was better in Bailey medium over BIII medium. The Bailey medium producedan average dry weight mass of 20 mg±10 mg, while the BIII mediumproduced an average dry weight mass of 16 mg±5 mg. It is understood,however, that certain types of fungi may grow better in certain types ofmedia. Accordingly, the limitation of the present invention to one typeof media over another is not necessary.

Oxygen

Fungal cultures were grown with or without oxygen-enhancement, i.e.,oxygen flush, to determine the effects of oxygen upon fungal growth.Fungal cultures were inoculated in Bailey media or BIII media, and grownon malt extract agar plates. From the plates, four agar disks (9 mm) ofcomplete fungal growth were removed and inoculated into 25-ml growthmedium in 125-ml flasks. The flasks were grown with and without oxygenflush, and were kept stationary at 27° C. and 70% RH for 21 days.Oxygen-enhanced cultures were flushed with oxygen every other day for aninterval of 20 seconds. After 21 days, the fungal mycelia wereharvested, air dried and measure for dry weight.

From the mycelia dry weight measurements, the results showed thatoxygen-enhanced incubation provided fungal growth similar to that of thewhite-rot fungi, Phanaerochytes chrysoporium (Kirk, et al. 1986). Asseen in Table 3 below, the enhanced growth effect has consequentlyproduced a lower pH value in the culture flasks, thus indicatingconfluent growth.

TABLE 3 Oxygen Effect on Fungal Growth with Two Defined Media Dryweights, Strain Medium O₂ mg (±S.D.) pH TFFH 294 Bailey + 23 ± 1 2.73 −20 ± 1 3.04 BIII + 21 ± 2 2.96 − 18 ± 2 3.61

Example 3

Nutrient Supplements: Corn Steep Liquor

Fungal inoculum containing Meruliporia incrassata (TFFH-294) was addedto malt extract liquid media supplemented with 1, 2.5 and 5% of sterilecorn steep liquor (CSL)(ADM corn processing, Cedar Rapids, Iowa). Mediacontaining no CSL was used as a control. Cultures were grown at 27° C.and 70% RH for 30 days. After 30 days, Mycelium was separated fromliquid culture by filtration, oven dried and weighed. The % dry weightwas determined as percent of the dry weight for the inoculum withoutCSL.

The addition of the corn steep liquor to the growth medium had aprofound effect on fungal growth. Various concentrations of CSL weretested and results are shown in Table 4 below. The highest enhancementeffect was obtained by adding 1% CSL in malt extract medium. Thisenhancement should limit the disadvantage experienced by various fungalstrains, such as TFFH-294 which are typically disadvantaged by theirslow growth characteristics that limits their competition with otherdominant fungi in nature, such as Postia placenta and Gloeophyllumtrabeum. Accordingly, the supplementation with a CSL nutrient will allowbetter competition in nature.

TABLE 4 Effect of CSL on TFFH-294 Growth CSL concentration % Dry weightgain 0 100 1.0% 321 2.5% 256 5.0% 196

Example 4

Effect of Corn Steep Liquor on Fungal Growth For Fungi Tolerant toDifferent Wood Chemical Preservatives

Fungal inoculum containing fungal strains exhibiting tolerance to eitherCCA, ACQ, creosote or pentachlorophenol were supplemented with 1%sterile CSL (ADM corn processing, Cedar Rapids, Iowa) in malt extractmedia to determine the effect of the CSL on the growth patterns of theseparate strains. Media without CSL was used as a control and consideredto exhibit 100% growth. Weight was determined from fungal myceliaincubated for 3 weeks in liquid malt extract, with or without CSL, at27° C. and 70% RH. After 3 weeks, the mycelia was removed from theliquid by filtration on filter paper, oven dried and weighed. The % dryweight was determined as percent of the dry weight for the inoculumwithout CSL. Table 5 illustrates the increase growth effect that the CSLprovides to the various strains used.

TABLE 5 Effect of corn steep liquor (CSL) on fungal growth in liquidmedium. Strain* CSL Net weight (g) % growth TFFH-294 − − 0.053 ± 0.011100 + 0.108 ± 0.027 203 FP-90848-T − − 0.079 ± 0.013 100 + 0.116 ± 0.023147 Mad-534 − − 0.049 ± 0.004 100 + 0.114 ± 0.006 233 Mad-617 − − 0.028± 0.003 100 + 0.106 ± 0.022 379 FP-103272-sp − − 0.058 ± 0.009 100 +0.114 ± 0.017 196 *TFFH-294 and FP-90848-T are CCA-tolerant andACQ-tolerant; Mad-534 and Mad-617 are creosote-tolerant; andFP-103272-sp is creosote-tolerant and penta-tolerant. Weight determinedfrom fungal mycelia incubated for 3 weeks in liquid malt extract, withor without cornstarch liquor (CSL), removed from liquid by filtration onfilter paper, oven dried and weighed. Medium without CSL (− −) is thecontrol, with 100% growth. Growth of medium with CSL (+) is expressed as% of the control.

Example 5

Perservative Wood Degradation Study

A degradation study was performed using fungal strains exhibitingtolerance to either CCA, ACQ, creosote or pentachlorophenol. Blocks ofwood were cut (1×1×0.3 inches) from southern pine (Pinus sp.) andtreated to CCA, ACQ, creosote and Creosotechlorophenol according toAmerican Wood Preserver's Association (AWPA) standards (“American WoodPreserver's Association: Book of Standards,” American Wood-Preserver'sAssociation, Woodstock, Md., 1991). The blocks of wood were sterilizedand allowed to cool.

Degradation of the treated wood block was observed by placing thesterile block on the surface of the fungal inoculum in the glass bottle.Fungi strains known to be tolerant to a specific preservative wereexposed to blocks having their respective preservative. The blocks werethen incubated for 10 weeks at 27° C. and 70% RH. After 10 weeks, theremaining portion of the blocks were removed and their dry weight losswas measured according to ASTM standards.

Table 6 below depicts the ability of the fungal inoculum having certainfungal strains to degrade the preservative-treated wood. As can be seenthe fungal strains of Meruliporia incrassata (TFFH-294), Antrodiaradiculosa (MJL-630), Meruliporia incrassata (Mad-563) and Antrodiaradiculosa (FP-90848-T) were able to degrade the Creosote-treated woodat an average of greater than 20% of the original dry weight of thewood. Antrodia radiculosa (FP-90848-T) was also able to degradeACQ-treated wood at an average of 29.9% of the original dry weight.Antrodia radiculosa strains FP-103272-sp, L-11659-sp, and FP-90848-T,and Meruliporia incrassata (Mad-563) were able to degrade on averageapproximately 3% of the wood block having a concentration ofpentachlorophenol. Finally, Antrodia radiculosa strains FP-103272-sp,L-11659-sp, and FP-10539-R, and Neolentinus lepideus (Mad-534) exhibitedthe ability to reduce the dry weight of wood having creosote by anaverage of approximately 3%.

TABLE 6 Fungal Degradation of Preservative-Treated Wood* UNTREATED ACQCCA Penta Creosote Fungus Avg SD Avg SD Avg SD Avg SD Avg SD Meruliporiaincrassata (TFFH-294) 62.2 2.9 9.7 5.7 36.8 2.7 1.9 0.3 1.8 0.2 Antrodiaradiculosa (MJL-630) 32.6 4.8 6.7 6.8 26.6 2.9 1.5 0.1 1.7 0.2Meruliporia incrassata (Mad-563) 62.5 2.5 3.5 0.1 23.7 7 4.1 2.5 1.5 0Antrodia radiculosa (FP-90848-T) 39.5 4.1 29.9 14.3 20.1 7.7 2.6 0.5 2.10.2 Antrodia radiculosa (FP-103272-sp) 24.6 6 0.7 0.1 6.5 4.7 4.7 2.35.5 2 Antrodia radiculosa (FP-105309-R) 27.2 3 4.4 4 2.3 0.8 2.4 0.6 2.90.8 Antrodia radiculosa (L-11659-sp) 23.1 2.7 0.8 0.3 1.3 1.3 5.3 1.83.2 1.8 Neolentinus lepideus (Mad-534) 38.8 5.3 1.4 0.3 −0.7 0.4 1.5 0.14.1 0.7 *Standard Method of Testing Wood Preservatives by LaboratorySoil-Block Cultures, ASTM D-1413-76

Effect of Additives On Degradation of Preservative-Treated Wood

A study was also performed to determine what effect the additives had onthe ability of certain fungal strains to degrade preservative-treatedwood. A fungal inoculum was prepared for each strain using one of theselected fungi as described above. First, the fungus was grown on 10 mlmalt extract agar in a glass bottle (2×2×5 h inches) and incubated at27° C., 70% RH, for 1-2 weeks until a confluent growth on the agar layeroccurred. A mixture of soft wood and hard wood sawdust was sterilizedand set for use as a lignocellulosic substrate.

Separate fungal inocula were prepared containing a nutrient supplementof either corn steep liquor or cornmeal and wheatbran. For the inoculumcontaining corn steep liquor, 10 g of the sterile sawdust was mixed with20 ml of sterile water containing 1% commercial corn steep liquor (v/v)and added to the glass bottle containing the fungal growth. For theinoculum containing cornmeal and wheatbran, 10 g of the sterile sawdustwas combined with the corn meal and wheat bran at 2.5% (w/w, 0.25 g cornmeal or wheat bran/10 g sawdust) for each ingredient, followed by 20 mlof sterile water. The inoculum was then added to another glass bottlecontaining the fungal growth. Incubation was continued in a stationarycondition and at a temperature of 27° C. and 70% RH. The length ofincubation depended on the rate of fungal growth, however, after about4-6 weeks of incubation fungal mycelia growth was obvious. The fungalstrains utilized are those depicted in table 7. These results indicatethat additives enhance degradation of preservative-treated wood.

TABLE 7 Effect of additives to degradation of preservative-treated wood.CHEMICAL FUNGAL SPECIES ISOLATE # WEIGHT* cmwb** CSL*** SYP**** CCAAntrodia radiculosa L-11659-sp 32.0 mg 150% 446% 972% Polyporus spFP134933 12.3 mg 1040%  577% 1284%  (unknown) F43G  1.0 mg 3500%  500%114% Diplomitoporus lindbladii FP134600 13.0 mg 469% 238% 753%Meruliporia incrassata TFFH-294 34.0 mg  32% 208% 179% ACQ Chainchlamydospore ME681 27.0 mg  63%  56% 248% Antrodia radiculosaFP-90848-T 17.0 mg 176% 188% 243% — UpK 38.0 mg 118% 103%  34% — UpL50.0 mg  82% 120% 678% Creosote Gloeophyllum Subferrugineum FPL 508 64.0mg 103%  39% Melanoporia niger MD278 66.0 mg  95% 127% 772% — UpK 51.0mg 112% 110%  14% Polyporus sp. FP101605 61.0 mg 101% 111% 224% *Weightof preservative-treated pine sawdust. Control weight. Growth is based on% of control weight. **% growth on pine sawdust amended with cornmeal(cm) and wheatbran (wb) ***% growth on pine sawdust amended withcornstarch liquor (SCL) ****SYP = Southern yellow pine sawdust with nopreservatives.

Example 6

Scale-up of Fungal Inoculum in Laboratory

Fungal inoculum containing Meruliporia incrassata (TFFH-294) wasprepared according to the present invention and utilized to determinethe effectiveness of the fungal inoculum on a larger scale. Although thefungal incoculum employed was directed towards wood treated with CCA, itis anticipated that similar results would be obtained on wood treatedwith creosote using creosote-tolerant fungal strains.

Fungal Culture Preparation

Five to seven Petri dishes (14 cm diameter) containing malt agar wereused to grow fungal inoculum containing the fungal strain Meruliporiaincrassata (TFFH-294). The Petri dishes were incubated at 27° C. and 70%RH until a confluent growth occurred on the agar layer. Agar chunks of1-1.5 inches square were removed and immediately transferred to thesolid substrate matrix already prepared as described below.

Solid Substrate Matrix in Tray

Solid substrate comprising a lignocellulosic substrate and nutrientsupplement were prepared in an aluminum tray (9×13×2.5 h inches). First,350 gm of sawdust was placed in the empty tray and the tray and sawdustautoclaved and cooled to room temperature. Once the tray and sawdust wascooled, 700 ml of sterile water having a 1% concentration of corn steepliquor was added and mixed to achieve a homogenous solid matrix. Thefungal squares were then gently mixed with the solid matrix, coveredwith foil and incubated. Incubation was in dark conditions and at 27°C., 70% RH, for a period of 4-8 weeks, or until a confluent growth wasobtained. After the fungus had reached confluent growth, the fungalinoculum was stored at 4° C. to prevent overgrowth.

Preservative Lumber Degradation in Degradation Chamber

Several large metal degradation chambers (33×6×8 h inches) with slidingcovers were custom-made for the lumber degradation study. A 2 inch layerof moistened soil having a water content of 35% was placed in thedegradation chamber. Formed pieces of 12 inch cut CCA-treated 2×4 lumberwas pieced on top of the soil and the metal degradation chambers weresterilized in an autoclave. After the chambers had cooled down to roomtemperature, the TFFH-294 fungal chips inoculum was poured onto thepreservative-treated lumber until the lumber was completely covered. Thechamber containing the inoculated lumber was then stored at 27° C. and70% RH for 12 weeks. At the end of the 12 week incubation period thewood was harvested and measured for dry weight loss to determine thelevel of degradation. Results showed a 28% degradation of CCA-treatedwood as compared to nontreated control wood.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions, that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims:

We claim:
 1. A method for bioremediating wood containing creosotecomprising the steps of: inoculating wood containing creosote with afungal inoculum comprising at least one creosote-tolerant fungusselected from the group consisting of Antrodia radiculosa(FP-103272-sp), Antrodia radiculosa (FP-105309-R), Antrodia radiculosa(L-11659-sp) and Neolentineus lepideus (Mad-534), at least onelignocellulosic substrate and at least one nutrient supplement, thelignocellulosic substrate and the nutrient supplement in an amountsufficient to produce a biomass of the fungal inoculum sufficient to atleast partially remediate the creosote; and aerating and hydrating theinoculated wood for a time and under conditions sufficient to allow thefungal inoculum to remediate the creosote.
 2. The method of claim 1wherein the creosote-tolerant fungus is Antrodia radiculosa(FP-103272-sp).
 3. The method of claim 1 wherein the creosote-tolerantfungus is Neolentinus lepideus (Mad-534).
 4. The method of claim 1wherein the lignocellulosic substrate is selected from the groupconsisting of sawdust, wood chips, rice straw, corn stalks, and wheatstraw.
 5. The method of claim 1 wherein the nutrient supplement isselected from the group consisting of corn steep liquor, cornmeal andwheatbran.
 6. The method of claim 1 wherein the inoculated wood isaerated and hydrated in dark, aerobic conditions, at a relative humidityof about 70%, and in a temperature range from about 20° C. to 35° C. 7.The method of claim 6 wherein the inoculated wood is aerated andhydrated in a temperature range from about 27° C. to 32° C.
 8. A methodfor degrading wood containing creosote comprising the steps of:inoculating wood containing creosote with a fungal inoculum comprisingat least one creosote-tolerant fungus selected from the group consistingof Antrodia radiculosa (FP-103272-sp), Antrodia radiculosa(FP-105309-R), Antrodia radiculosa (L-11659-sp) and Neolentineuslepideus (Mad-534), at least one lignocellulosic substrate and at leastone nutrient supplement, the lignocellulosic substrate and nutrientsupplement in an amount sufficient to produce a biomass of the fungalinoculum sufficient to degrade the wood; and aerating and hydrating theinoculated wood for a time and under conditions sufficient to allow thefungal inoculum to degrade the wood to reach a degradation product. 9.The method of claim 8 wherein the creosote-tolerant fungus is Antrodiaradiculosa (FP-103272-sp).
 10. The method of claim 8 wherein thecreosote-tolerant fungus is Neolentinus lepideus (Mad-534).
 11. Themethod of claim 8 wherein the lignocellulosic substrate is selected fromthe group consisting of sawdust, wood chips, rice straw, corn stalks,and wheat straw.
 12. The method of claim 8 wherein the nutrientsupplement is selected from the group consisting of corn steep liquor,cornmeal and wheatbran.
 13. The method of claim 8 wherein the inoculatedwood is aerated and hydrated in dark, aerobic conditions, at a relativehumidity of about 70%, and in a temperature range from about 20° C. to35° C.
 14. The method of claim 13 wherein the inoculated wood is aeratedand hydrated in temperature rage from about 27° C. to 32° C.
 15. Themethod of claim 8, wherein the degradation product exhibits a loss indry weight of an amount in excess of 2.5% of the dry weight of the wood.16. A fungal inoculum for degrading and bioremediating wood containingcreosote comprising at least one creosote-tolerant fungus selected fromthe group consisting of Antrodia radiculosa (FP-103272-sp), Antrodiaradiculosa (FP-105309-R), and Antrodia radiculosa (L-11659-sp) andNeolentinus lepideus (Mad-534), at least one lignocellulosic substrate,and at least one nutrient supplement.
 17. The fungal inoculum of claim16 wherein the creosote-tolerant fungus is Antrodia radiculosa(FP-103272-sp).
 18. The fungal inoculum of claim 16 wherein thecreosote-tolerant fungus is Neolentinus lepideus (Mad-534).
 19. Thefungal inoculum of claim 16 wherein the lignocellulosic substrate isselected from the group consisting of sawdust, wood chips, rice straw,corn stalks, and wheat straw.
 20. The fungal inoculum of claim 16wherein the nutrient supplement is selected from the group consisting ofcorn steep liquor, cornmeal and wheatbran.